Worldwide, the most important cause of bronchiolitis and serious lower respiratory tract disease in infants and young children is respiratory syncytial virus (RSV) (Collins et al., “Respiratory syncytial virus,” IN: Fields B, Knipe, D., Howley P., eds. Fields Virology. 3rd ed. 1. Philadelphia: Lippencott-Raven Publishers:1313 (1996); Crowe, J. E., Jr., “Current approaches to the development of vaccines against disease caused by RSV and parainfluenza virus (PIV),” Meeting Report, WHO Programme for Vaccine Development. Vaccine 13:415 (1995); and Welliver et al., “Clinical and laboratory diagnosis of respiratory syncytial virus infection,” Critical Reviews in Clinical Laboratory Sciences 13:213 (1981)). Natural infection with RSV provides limited protection from reinfection and disease as demonstrated by recurrence of severe RSV infections throughout life (Hall et al., “Immunity to and frequency of reinfection with respiratory syncytial virus,” J. Infect. Dis. 163:693 (1991); and Henderson et al., “Respiratory syncytial virus infections, reinfections and immunity: a prospective. Longitudinal study in young children,” N. Engl. J. Med. 300:530 (1979)). Moreover, the peak incidences of serious RSV disease are in young infants who have maternally-acquired neutralizing RSV antibodies. Thus, the development of RSV vaccines has long been a priority for infants and young children (Crowe, J. E. Jr., “Current approaches to the development of vaccines against disease caused by respiratory syncytial virus (RSV) and parainfluenza virus (PIV). A meeting report of the who programme for vaccine development,” Vaccine 13:415 (1995); “New vaccine development: establishing priorities; diseases of importance in the United States,” Washington D.C., Institute of Medicine National Academy Press (1985); “New vaccine development: establishing priorities, diseases of importance in developing countries,” Washington D.C. National Academy Press II (1986); Doherty P. C., “Vaccines and cytokine-mediated pathology in RSV infection,” Trends in Microbiology 2:148 (1994); and Murphy et al., “Current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses,” Virus Research 11:1 (1988)). More recently, vaccines are being developed for older children at risk for serious complications of infection and for the elderly (Piedra et al., “Purified fusion protein vaccine protects against lower respiratory tract illness during respiratory syncytial virus season in children with cystic fibrosis,” Pediatric Infectious Disease Journal 15:23 (1996); Han et al., “Respiratory syncytial virus pneumonia among the elderly: an assessment of disease burden,” Journal of Infectious Diseases 179:25 (1999); and Falsey et al., “Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in ambulatory adults over age 60,” Vaccine 14:1214 (1996)).
RSV vaccine development has been focused toward live, temperature sensitive, attenuated vaccines and subunit vaccines based on the F and/or G glycoproteins (Groothuis et al., “Safety and Immunogenicity of a purified F protein respiratory syncytial virus (PFP-2) vaccine in seropositive children with bronchopulmonary dysplasia,” Journal of Infectious Diseases 177:467 (1998); Neuzil et al., “Adjuvants influence the quantitative and qualitative immune response in BALB/c mice immunized with respiratory syncytial virus FG subunit vaccine,” Vaccine 15:525 (1997); and Siegrist et al., “Protective efficacy against respiratory syncytial virus following murine neonatal immunization with BBG2NA vaccine: influence of adjuvants and maternal antibodies,” Journal of Infectious Diseases 179:1326 (1999)). In animal model systems, the F glycoprotein is generally the most effective with the G glycoprotein being the next most effective in inducing neutralizing antibodies and protective immunity. Vaccination with the G glycoprotein, however, generally induces only limited protection (Stott et al., “Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus,” J. Virol. 61:3855 (1987); and Connors et al., “Respiratory syncytial virus (RSV) F, G, M2 (22K), and N proteins each induce resistance to RSV challenge, but resistance induced by M2 and N proteins is relatively short-lived,” Journal of Virology 65:1634 (1991)). In clinical studies, no vaccine has yet proven safe and efficacious, mainly because candidate live virus vaccines have been under or over attenuated or have reverted to wildtype phenotype (Karron et al., “Evaluation of two live, cold-passaged, temperature-sensitive respiratory syncytial virus vaccines in chimpanzees and in human adults, infants, and children,” Journal of Infectious Diseases 176:1428 (1997)). However, the recent availability of an RSV infectious clone has expanded the options in developing live virus candidate vaccine strains, and renewed hope that a safe and efficacious live virus vaccine can be developed (Teng et al., “Altered growth characteristics of recombinant respiratory syncytial viruses which do not produce NS2 Protein,” Journal of Virology 73:466 (1999); Skiadopoulos et al., “Attenuation of the recombinant human parainfluenza virus type 3 CP45 candidate vaccine virus is augmented by importation of the respiratory syncytial virus CPTS530 L polymerase mutation,” Virology 260:125 (1999); and Whitehead et al., “Addition of a missense mutation present in the 1 gene of respiratory syncytial virus (RSV) CPTS530/1030 to RSV vaccine candidate CPTS248/404 increases its attenuation and temperature sensitivity,” Journal of Virology 73:871 (1999)). Nevertheless, there remains a need for efficacious methods and adjuvants for enhancing the safety and efficacy of RSV vaccines.
Development of subunit vaccines, in particular, has been hindered by concerns about the enhanced lung disease that occurred in young children who received a formalin-inactivated RSV (FI-RSV) vaccine, and then had natural RSV infection (Chin et al., “Field evaluation of a respiratory syncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population,” Am J Epidemiol 89:449 (1969)). Although the reasons for this vaccine-augmented disease remain uncertain, evidence in the BALB/c mouse model suggests that a heightened Th2 cytokine response may in part be responsible (Anderson et al., “Cytokine Response to respiratory syncytial virus stimulation of human peripheral blood mononuclear cells,” Journal of Infectious Diseases 170:1201 (1994); Connors et al., “Enhanced pulmonary histopathology induced by respiratory syncytial virus (RSV) challenge of formalin-inactivated RSV-immunized BALB/c mice is abrogated by depletion of interleukin-4 (IL-4) and IL-10,” Journal of Virology 68:5321 (1994); and Waris et al., “Respiratory synctial virus infection in BALB/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant TH2-like cytokine pattern,” Journal of Virology 70:2852 (1996)). In these studies, enhanced lung pathology in FI-RSV vaccinated mice challenged with live RSV was shown to be mediated by Th2-type CD4+ T cells expressing IL-4, IL-5, IL-6, and IL-10. In contrast, mice immunized and challenged with live RSV do not develop extensive lung pathology and respond with a mixed Th1/Th2 immune response (Anderson et al. (1994); Graham et al., “Priming immunization determines T helper cytokine mRNA expression patterns in lungs of mice challenged with respiratory syncytial virus,” Journal of Immunology 151:2032 (1993); Hussell et al., “Th1 and Th2 cytokine induction in pulmonary T cells during infection with respiratory syncytial virus,” Journal of General Virology 77:2447 (1996); and Srikiatkhachom et al., “Virus-specific memory and effector T lymphocytes exhibit different cytokine responses to antigens during experimental murine respiratory syncytial virus infection,” Journal of Virology 71:678 (1997)). Based on this possible role of a heightened Th2 cytokine response in enhancing lung disease upon live RSV infection, there is a need for methods and/or reagents for promoting a Th1 response over a Th2 response to live viral challenge.
Since passive administration of high titered neutralizing RSV antibodies can decrease the risk of serious RSV disease, one of the best indicators of protection from RSV disease is high titers of neutralizing antibodies (Groothuis et al., “Use of intravenous gamma globulin to passively immunize high-risk children against respiratory syncytial virus: safety and pharmacokinetics. The RSVIG study group,” Antimicrobial Agents & Chemotherapy 35:1469 (1991); and Hemming et al., “Hyperimmune globulins in prevention and treatment of respiratory syncytial virus infections,” Clinical Microbiology Reviews 8:22 (1995)). Unfortunately, both attenuated live and subunit candidate vaccines induce only modest increases in antibodies (Dudas et al., “Respiratory syncytial virus vaccines,” Clinical Microbiology Reviews 11:430 (1998); and Murphy et al., “An update on approaches to the development of respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) vaccines,” Virus Research 32:13 (1994)). As evidenced by the variety of adjuvants that are being evaluated for their ability to enhance the immune response to subunit vaccines (See e.g., Xin et al., “Immunization of rantes expression plasmid with a DNA vaccine enhances HIV-1-specific Immunity,” Journal of Applied Biomaterials (Orlando) 92:90 (1999); Ciupitu et al., “Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes,” Journal of Experimental Medicine 187:685 (1998); and Hancock et al., “Formulation of the purified fusion protein of respiratory syncytial virus with the saponin QS-21 induces protective immune responses in BALB/c Mice that are similar to those generated by experimental infection,” Vaccine 13:391 (1995)), there remains a need for an adjuvant that is effective at enhancing immune responses to RSV vaccines, especially subunit vaccines.
One potential immune-enhancing molecule is CD40L which is important to productive interactions between T cells and antigen presenting cells (Armitage et al., “CD401: a multi-functional ligand,” Seminars in Immunology 5:401 (1993); Grewal et al., “The CD40 Ligand. At the center of the immune universe?” Immunologic Research 16:59 (1997); Laman et al., “Functions of CD40 and its ligand, GP39 (Cd401),” Critical Reviews in Immunology 16:59 (1996); Ramshaw et al., “Cytokines and immunity to viral infections,” Immunological Reviews 159:119 (1997); van Kooten et al., “Functional role of CD40 and its ligand,” International Archives of Allergy & Immunology 113:393 (1997); van Kooten et al., “Functions of CD40 on B cells, dendritic cells and other cells,” Current Opinion in Immunology 9:330 (1997); Bennett et al., “Help for a cytotoxic-T-cell response is mediated by CD40 signaling,” Nature 393:478 (1998); and Ridge et al., “A conditioned dendritic cell can be a temporal bridge between a CD4+T-helper and a T-killer,” Nature 393:474 (1998)). CD40L has been shown to enhance both humoral and cellular immune responses (Grewal et al. (1997); Laman (1996); Gurunathan et al., “CD40 ligand/trimer DNA enhances both humoral and cellular immune responses and induces protective immunity to infectious and tumor challenge,” Journal of Immunology 161:4563 (1998); and Durie et al., “The role of CD40 in the regulation of humoral and cell-mediated immunity,” Immunology Today 15:406 (1994); Klaus et al., “CD40 and its ligand in the regulation of humoral immunity,” Seminars in Immunology 6:279 (1994); and Noelle et al., “CD40 and its ligand in cell-mediated immunity,” Agents & Actions—Supplements 49:17 (1998)). For example, CD40L expression has been shown to enhance T cell and APC activation and signaling, the importance of which has been revealed by studies of CD40L−/− mice (Borrow et al., “CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8+ CTL response,” Journal of Experimental Medicine 183:2129 (1996); Castigli et al., “CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation,” Proceedings of the National Academy of Sciences of the United States of America 91:12135 (1994); Cosyns et al., “Requirement of CD40-CD40 ligand interaction for elimination of Cryptosporidium parvum from mice,” Infection & Immunity 66:603 (1998); Grewal et al., “Impairment of antigen-specific T-cell priming in mice lacking CD40 ligand,” Nature 378:617 (1995); Kawabe et al., “The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation,” Immunity 1:167 (1994); Kennedy et al., “CD40/CD40 ligand interactions are required for T cell-dependent production of interleukin-12 by mouse macrophages,” European Journal of Immunology 26:370 (1996); Lei et al., “Disruption of antigen-induced inflammatory responses in CD40 ligand knockout mice,” Journal of Clinical Investigation 101:1342 (1998); Soong et al., “Disruption of CD40-CD40 ligand interactions results in an enhanced susceptibility to Leishmania amazonensis infection,” Immunity 4:263 (1996); and Stout et al., “Impaired T cell-mediated macrophage activation in CD40 ligand-deficient mice,” Journal of Immunology 156:8 (1996)) and in CD40L−/− humans (Allen et al., “CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome,” Science 259:990 (1993)). Coincident expression of CD40L has been shown to promote T cell mediated immunity (Ramshaw et al. (1997); Yang et al., “Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung tissues,” Journal of Virology 70:6370 (1996); Ruby et al., “CD40 ligand has potent antiviral activity,” Nature Medicine 1:437 (1995); Couderc et al., “Enhancement of antitumor immunity by expression of CD70 (CD27 ligand) or CD154 (CD40 ligand) costimulatory molecules in tumor cells,” Cancer Gene Therapy 5:163 (1998); and Brown et al., “Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice,” Nature Medicine 4:1253 (1998)). Administering anti-CD40 monoclonal antibody to mice together with pneumococcal polysaccharide generated strong, isotype-switched antibody responses (Dullforce et al., “Enhancement of T cell-independent immune responses in vivo by CD40 antibodies,” Nature Medicine 4:88 (1998)). Immunization of BALB/c mice with DNA plasmids expressing β-galactosidase and a protein consisting of CD40L linked to a leucine zipper motif increased the Th1-type immune response to β-galactosidase (Gurunathan et al. (1998)). The importance of CD40L expression in the development of Th1 cytokine responses was also demonstrated by antibody inhibition studies in which anti-CD40L antibody decreased Th1-mediated autoimmune diabetes in NOD mice by reducing IL-12 secretion and slightly increasing IL-4 production (Balasa et al., “CD40 ligand-CD40 interactions are necessary for the initiation of insulitis and diabetes in nonobese diabetic mice,” Journal of Immunology 159:4620 (1997)).
Several studies that utilized CD40L deficient mice reveal a role for CD40L in both humoral and cellular immune responses to viruses. Constitutive retroviral expression of CD40L restored antigen-specific cytolytic and humoral immune responses in CD40L−/− mice infected intranasally with the HKx31 strain of influenza (Brown et al. (1998)). Infection of mice with recombinant vaccinia virus that expressed CD40L markedly enhanced viral clearance by both an IFNγ-dependent mechanism and a novel CD40L-dependent mechanism (Ruby et al. (1995)). In studies that examined the anti-adenovirus response in CD40L−/− mice, diminished CD4+ T cell priming and reduced IL4, IL-10 and IFNγ cytokine expression occurred in the absence of CD40L (Yang et al. (1996)). A role for CD40L in antiviral B and T cell immune responses was also shown in CD40L−/− mice challenged with lymphocytic choriomeningitis virus (LCMV), (Borrow et al. (1996); Oxenius et al., “CD40-CD40 ligand interactions are critical in T-B cooperation but not for other anti-viral CD4+ T cell functions,” Journal of Experimental Medicine 183:2209 (1996)). CD40L−/− mice infected with LCMV were capable of generating primary CTL responses, but had defective memory CTL responses. Furthermore, primary anti-LCMV IgG1 antibody responses were severely impaired in the absence of CD40L. Although an involvement of CD40L in immune responses has been established, it has not been established or suggested that CD40L, or vectors capable of expressing CD40L, are effective in enhancing an immune response, especially a Th1 cytokine immune response, to RSV.
Li et al. (“Nucleic Acid Respiratory Syncytial Virus Vaccines,” U.S. Pat. Nos. 5,880,104 (1999) and 5,843,913 (1998)) describe vaccines for RSV that utilize the RSV F protein expressed from the cytomegalovirus promoter.
Brams et al. (“Neutralizing High Affinity Human Monoclonal Antibodies Specific to RSV F-Protein and Methods for their Manufacture and Therapeutic Use Thereof,” U.S. Pat. Nos. 5,939,068 (1999) and 5,811,524 (1998)) describe methods for producing antibodies against the RSV F protein, and describe several antibodies developed using these methods.
Gurunathan et al., (“CD40 Ligand/Trimer DNA Enhances Both Humoral and Cellular Immune Response and Induces Protective Immunity to Infectious and Tumor Challenge,” J. Immunol. 161, 4563 (1998)), describes the use of plasmids encoding a protein containing the CD40L coding region fused to a leucine zipper motif, called the CD40 ligand/trimer, to enhance Th1 and cytotoxic T-lymphocyte responses.
Alderson et al., (“Method for Stimulating an Immune Response,” WO96/26735 (1996)), describes the use of a soluble, trimer-forming fragment of CD40L to enhance production of interleukin 12 in vitro, and to treat infection with Leishmania or Pneumocystis in CD40L-deficient mice.
The current invention provides adjuvants and methods that utilize a source of a CD40 binding protein, such as a vector operatively linked to CD40L, to enhance an immune response, especially a Th1 cytokine immune response, to an RSV vaccine.